The analysis of both environmentally relevant substances and natural products has, to a large extent, moved on from mass spectrometry/gas chromatography (GC-MS) to the coupling of mass spectrometry with liquid chromatography (LC-MS) or capillary electrophoresis (CE-MS). The reasons for this are manifold: on the one hand, most of the substances in this field currently under investigation can no longer be vaporized without decomposition, an indispensable requirement for gas chromatography; on the other, these substances are usually already in an aqueous state at the locations where they occur. In this description, the vaporization capability forms the border between the “light molecules” and the “medium weight molecules”.
“Environmentally relevant substances” should be understood here as such substances and their decomposition products (metabolites) which we encounter in our environment—nature, plants, animals—in mainly aqueous solutions where they are subject to continuous chemical, enzymatic or microbiological decomposition. This could be the metabolism of potential pharmaceuticals or the decomposition of herbicides or pesticides in humans, animals, plants, nature, or even as residues in food. The original substances are usually man-made and could possibly be known at the beginning of the analysis, though this is not necessary.
“Natural products” are defined here as the large group of organic substances which occur in the untouched nature, especially in animals and plants but also in fossil deposits, and which do not belong to the group of chain biopolymers (proteins, DNA, polysaccharides) termed “high-molecular”. These natural products include many hormones, vitamins, and active substances in plants as well as the infinitely large number of ingredients in crude oils and coals. In addition to purely organic substances, metal-organic and mineral-organic substances also occasionally occur here.
Environmentally relevant and natural products, and the whole substance group of the medium weight range, are of great general interest. As briefly described above, a small proportion of them can be identified using GC-MS. This identification is relatively easy because the usually used electron impact ionization provides mass spectra which can be easily identified by library searches. The nowadays preferred method LC-MS, however, has a much more general application. It is separation by liquid chromatography (HPLC=high performance liquid chromatography) with subsequent ionization using electro spray ionization (ESI). This method offers advantages but also a series of difficulties, beginning with the fact that the spectra contain hardly any characteristic fragment ions. In order to overcome these difficulties, the spectra of positive and negative ions are acquired in quick succession and, in both cases, the daughter ion spectra of the prominent ions in each case are also automatically acquired; but, even then, rapid identification is problematic. This is because they belong to a large number of different chemical classes, behaving differently in fragmentation, and also because of the frequent formation of simple or complex adduct ions. The term “prominent ions” is taken here to mean ions whose intensity makes them stand out; depending on the affinity to adducts, these could be the pseudo-molecular (protonated) ions or they could also be some adduct ions. Up to now, there is no rapid recognition method for adduct ions.
Besides the separation of substances by liquid chromatography, separation using different types of capillary electrophoresis is also coming to the fore. The term “pseudo-molecular ions” is defined here as the protonated molecular ions (hydrogen ion adducts) in the mass spectra of the positive ions, and the deprotonated molecular ions (hydrogen ion deducts) in the mass spectra of the negative ions. Depending on the voltage polarity applied, the process of electro spray ionization creates either positive or negative ions which can be acquired as positive and negative ion mass spectra by appropriate mass spectrometers. The negative deprotonated ions probably arise as a result of the attachment of an OH− ion to the substance molecule, with the immediate splitting off of H2O.
The analyte substances under consideration here generally have molecular weights between around 100 and 1000 atomic mass units and are usually found in complex solutions which also contain varying degrees of salts and hence both cations and anions, mainly various alkali ions and chlorine ions which form adduct ions from substance molecules with these cations and anions. Electro spray ionization generates mainly singly charged ions but there are exceptions here, as well, (particularly in the case of heavier analyte substances) where doubly charged ions occur in part as adduct ions. The separation of mixtures using liquid chromatography and the subsequent electro spray ionization thus frequently generates alkali-adduct ions (cation adducts) of the form (M+Kat)+ in the positive mass spectrum instead of the (M+H)+ pseudo-molecular ions usually formed; in the case of negative ions, anion adducts of the form (M+An)− are frequently formed instead of the pseudo-molecular ions (M−H)−.
The affinity of the substances to the alkali ions varies drastically. There are substances which appear almost exclusively in the form (M+Na)+ in this type of analysis, i.e. only as adducts with sodium. The signal of the protonated pseudo-molecular ions (M+H)+ can thus be very small or even disappear completely in the background noise. It is therefore difficult to identify these substances, particularly since, as yet, the state of the art to acquire daughter ion spectra just by the search for prominent ions does not always include the acquisition of the daughter ion spectra of the pseudo-molecular ions. It is, however, possible that various adduct ions appear side by side, for example (M+Na)+ and (M+K)+.
An instrument to analyze environmentally relevant substances and natural products consists of the coupling of a liquid chromatograph via a device for electro spray ionization to a mass spectrometer which can measure both positive and negative ions and which possesses a device to fragment the ions in order to acquire the daughter ion spectra. A high-frequency ion trap mass spectrometer according to Wolfgang Paul is mentioned as one example of such a mass spectrometer. A second example is the Fourier transform mass spectrometer. Other types are tandem mass spectrometers consisting of quadruple filters and collision cells in connection with a second mass spectrometer, for example, a time-of-flight mass spectrometer with orthogonal ion injection.
A favorable method for identifying substances with such an instrument therefore consists of not only acquiring both positive and negative mass spectra for each eluting substance in rapid succession, but also the daughter ion spectra for both polarities. The type of parent ion chosen for acquiring the daughter ion spectra generally depends on the intensity of the ions in the mass spectrum, usually supported by a list of prohibited ions which forbids the use of the ions of impurities which are always present. In such cases, it is frequently only the daughter ion spectrum of an adduct ion which is acquired, since the pseudo-molecular ions are often only of low intensity. The daughter ion spectrum of the adduct ions, however, generally contain very little information, since they often indicate only the loss of the adduct and contain no further information concerning the structure of the substance. Mass spectra and daughter ion spectra are then used to identify the substance by a spectrum library, said spectrum library contains positive and negative substance spectra as well as daughter ion spectra of the pseudo-molecular ions and, where possible, daughter ion spectra of the most common adduct ions.
However, since the spectra generated by electro spray ionization do not, as a rule, contain any fragment ions, and since the daughter ion spectra of these substance groups also frequently contain relatively little information compared to electron impact spectra because they have only few fragment ions, the results of the identification thus obtained are ambiguous in the majority of analyses. As explained above, the daughter ion spectra of adduct ions, in particular, are frequently virtually useless for an identification.
Even though the mass spectra of the substances contain practically no fragment ions, they can be very complex. The process of electro spray ionization generates primarily singly charged ions but also doubly charged ones. In addition, ions of the substance dimers, in some cases even substance trimers, are formed. All these ions are subject to adduct formation: adducts of the singly charged molecular ions, the doubly charged molecular ions and the dimer ions. These adducts can, in turn, be single anion or cation adducts or also more complex adducts with several anions or cations. The type of adduct depends on those substances which are capable of dissociation, usually salts, which remain in the solution after sample preparation, and also on the affinity of the substances to the various anions and cations. It is generally difficult to remove the salts, in many cases it is nearly impossible. The substances capable of dissociation can, in turn, influence the formation of dimers and trimers. The requirements made of the solvent cleanliness frequently extend beyond the degree of purity of the solvents commercially available as standard.
Since the composition of the solution containing the substances is never completely the same, alone by virtue of the origin of the substances, it follows that the spectra, with their complex formation of adducts, dimer-adducts and doubly charged adducts are never similar enough to permit an unequivocal identification of the substance.